Sds loading buffer

For pCREB analysis, cells were cultured in serum free media for 6 hours, followed by a 15 minute treatment with 10 nM E2 or BPA. Protein extracts were prepared by homogenizing cells on ice in RIPA buffer containing protease inhibitors. Protein concentrations were measured using the Bradford method. Samples (40 μg/lane) were boiled at 95°C for 10 minutes in sodium dodecyl sulfate (SDS) loading buffer (Boston BioProducts, Ashland, MA, USA), separated on 8% SDS-PAGE gels and transferred to a polyvinylidenedifluoride membrane according to manufacturers’ protocols (Bio-Rad Laboratories, Hercules, CA, USA). After incubation with 5% BSA in TBST for 60 min, the membrane was incubated with primary antibodies at 4°C overnight. Membranes were washed three times for 10 minutes and incubated with horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies for 1 hour at room temperature. Blots were washed with TBST three times and developed with an ECL system (Thermo Fisher Scientific) according to the manufacturer’s protocols.

Protein extracts were prepared by homogenizing cells on ice in RIPA buffer containing protease inhibitors. Protein concentrations were measured using the Bradford method. 40 μg of each sample was boiled at 95°C for 10 minutes in sodium dodecyl sulfate (SDS) loading buffer (Boston BioProducts, Ashland, MA, USA), separated on 8% SDS-PAGE gels and transferred to a polyvinylidenedifluoride membrane using a transfer apparatus according to the manufacturer’s protocols (Bio-Rad Laboratories, Hercules, CA, USA). After incubation with 5% BSA in TBST for 60 min, the membrane was incubated with antibodies (NANOG (1:2,000) or β-Actin (1:5,000)) at 4°C overnight. Membranes were washed three times for 10 min and incubated with a 1:10,000 dilution of horseradish peroxidase-conjugated anti-rabbit or anti-mouse antibodies for one hour at room temperature. Blots were washed with TBST three times and developed with the ECL system (Thermo Fisher Scientific) according to the manufacturer’s protocols.